PROJECT SUMMARY Alcohol use disorder (AUD) has many risk factors, both environmental and genetic, and represents a complex behavioral trait producing profound biomedical and social consequences. Identifying genes that engender AUD risk and contribute to the underlying neurobiological mechanisms represents an important first step in designing effective treatments. Published and preliminary studies make it apparent that chloride intracellular channel 4 (Clic4) expression has a major influence on ethanol sensitivity, a major risk factor for alcohol dependence in humans. Recent unpublished data has also defined a role for Clic4 in determining ethanol preference and intake. Together, these studies underscore the potential impact of Clic4 on the molecular basis of AUD and validate further investigation. In the work proposed here, Clic4 will be deleted separately in neurons and oligodendrocytes in the prefrontal cortex (PFC) of mice and the cell type-specific roles of Clic4 in drinking behavior, ethanol sensitivity, and anxiety-like behavior will be evaluated. These deletions will be performed through stereotactic microinjection of AAV-Cre into adult Clic4-floxed mice. Behaviors will be evaluated using 3-bottle-choice intermittent ethanol access, loss of righting reflex, and the light/dark box test. In addition to describing the role of Clic4 in ethanol-related behavior, a novel and impactful characterization of its mechanisms will be conducted. CLIC4 has been shown to translocate to the nucleus and interact with transcription factors thereby altering gene expression. Studying the ethanol-Clic4 interactome by manipulating Clic4 in the presence of ethanol could reveal the mechanistic framework underlying its role in ethanol-related behavior. This potential will be evaluated by characterizing the transcriptomic changes in the PFC that follow ethanol exposure in mice deficient for Clic4 in neurons and oligodendrocytes. Cell type-specific deletions of Clic4 will be performed in adult mice using a tamoxifen-inducible Cre/loxP system. RNAseq will provide identification of differential gene expression and downstream bioinformatic analysis will identify enriched biological pathways and relevant gene networks. The studies defined in this proposal will characterize the extent which Clic4 modifies ethanol-related behavior and provide an important first step towards understanding the mechanism.